Milk clotting enzymes are widely used in the cheese manufacturing industry to provide a curd of the major milk proteins. Commercially available milk clotting enzymes include native (or homologous) enzymes derived from microbial species or animal tissue sources such as calf stomachs, or such enzymes can be provided as gene products of recombinant cells expressing a heterologous milk clotting enzyme of animal or microbial origin.
Milk clotting enzymes of microbial origin are in commercial use in the dairy industry. In the following, such enzymes are also referred to as microbial milk clotting enzymes, microbial rennets or microbial coagulants. Examples of such enzymes include proteases natively produced by the zygomycete filamentous fungal species Rhizomucor miehei and Rhizomucor pusillus and protease produced by the fungal species Endothia parasitica. Enzymes having milk clotting activity can also be produced by other microbial species, including Rhizopus species, Physarum species, Penicillium species and Bacillus species.
The major milk clotting enzyme derived from Rhizomucor miehei is an aspartic protease (EC 3.4.23) being produced extracellularly by this fungal species and having a relatively high milk clotting activity and a relatively low proteolytic activity, i.e. with a desirable low ratio between proteolytic activity and milk clotting activity. A commercial coagulant containing Rhizomucor miehei aspartic protease is also referred to in the industry as a Mucor rennin.
Microbial coagulant compositions may be based on enzymes produced by microbial strains naturally producing milk clotting enzymes (homologous enzymes), or they can be based on enzymes produced by a heterologous strain. Thus, as an example Aikawa et al. 1990, J. Biol. Chem., 265, 13955-13959 have disclosed the expression of the Rhizomucor pusillus rennin in a strain of Saccharomyces cerevisiae.
One advantage of using a heterologous production strain in the industrial manufacturing of microbial coagulants such as aspartic proteases is that undesirable contamination with other proteases generally occurring in homologously produced enzyme preparations can be at least partially eliminated.
It is well-known that microbially produced enzymes may be glycosylated when expressed, the degree of glycosylation depending on the type of enzyme and the microbial species expressing the enzyme. Thus, Aikawa et al., supra found that the mature Rhizomucor pusillus aspartic protease as produced homologously in that species only contained a few glycosidic moieties in its molecules whereas the enzyme, when it was expressed in Saccharomyces cerevisiae, was highly glycosylated (about 37 residues/mole). These investigators found that deglycosylation of the heterologous protease which was achieved by treating this milk clotting enzyme with endo-.beta.-N-acetylgalactosaminidase resulted in enhancement of its milk clotting activity, whereas treating the homologously produced protease similarly did not affect the milk clotting activity hereof.
Gray et al., 1986, Gene, 48, 41-53 disclosed expression of Rhizomucor miehei aspartic protease in Aspergillus nidulans and found that this heterologously produced protease had the same specific activity as determined in a milk turbidity assay as had the corresponding homologously produced protease.
Recently, a commercial Rhizomucor miehei derived aspartic protease produced in Aspergillus oryzae has been disclosed (Novo Nordisk, Biotimes, June 1994). The milk clotting activity of this heterologous protease was studied by Christensen et al., 1988, Biotechnology, 6, 1419-1422. These authors found that the protease was overglycosylated which, however, according to these authors did not alter the specific activity of the enzyme. In WO 94/24880 it is specifically disclosed that this recombinant aspartic protease has an N-bound glucosamin, galactose and mannose content of 100% more than the corresponding native enzyme.
The above protease is marketed by Novo Nordisk under the trade name Novoren.RTM.. In the documentary material which was filed by the manufacturer with the U.S. Food and Drug Administration to obtain approval of its recombinant aspartic protease there are referred to experiments where the enzyme was treated with endoglycosidase H (Endo-H) which caused deglycosylation. It is stated that the extra glycosylation of the heterologously expressed enzyme does not alter the specific activity of the enzyme significantly. Furthermore, in the above Novo Nordisk publication, Biotimes, June 1994 it is stated on page 2 about Novoren.RTM. that the manufacturer cannot see how this enzyme preparation can be further improved.
McBride-Warren et al. 1973, Biochimica et Biophysica Acta, 328, 52-60 disclosed that homologous Rhizomucor miehei aspartic protease was glycosylated and that the removal of 40-50% of the sugar moieties (deglycosylation) resulted in a loss of up to 50% of the enzymatic activity.
Homologous aspartic protease produced by Rhizomucor miehei has been widely used in the dairy industry as a milk clotting enzyme for about 25 years. Examples of commercial products containing this enzyme are Rennilase.RTM. marketed by Novo Nordisk and the commercial products Hannilase.RTM. and Modilase S.RTM. sold by Chr. Hansen A/S.
It has now surprisingly been found that homologous Rhizomucor miehei aspartic protease, contrary to what has been stated in the prior art, acquires a significantly enhanced milk clotting activity when it is deglycosylated, and furthermore, that the milk clotting activity of heterologous Rhizomucor miehei aspartic protease as produced in Aspergillus oryzae, contrary to the above disclosures by a manufacturer hereof, is enhanced significantly by deglycosylation.